This project will target the essential RNA editing multi-protein complex (the editosome) in related trypanosomatid pathogens (Trypanosoma brucei, T. cruzi, and Leishmania spp.), which are the causative agents of African sleeping sickness, Chagas' disease, and leishmaniasis, respectively. It will identify inhibitors of this essential process, utilizing the capacity of a FRET-based assay for a sensitive ?mix and measure? in vitro monitoring of RNA editing activity in T. brucei, which we developed in a previous R21 assay development grant. Our preliminary data show that the editosomes are highly conserved among trypanosomatids; these data suggest the important possibility that inhibitors found against T. brucei editosome proteins will also be effective against the editosome in related trypanosomatid pathogens (T. cruzi and Leishmania spp.). To address the assay feasibility, we completed a pilot screen of the library of pharmacologically active compounds (LOPAC1280) and discovered new inhibitors that specifically interfere with RNA editing and kill T. brucei in vitro. Motivated by these recent experiments, we propose to screen a large library of compounds against the editosome in collaboration with Conrad Prebys Center for Chemical Genomics at Sanford Burnham Prebys Medical Discovery Institute. In Aim 1, editosomes and RNAs will be prepared, while we further miniaturize to 1536-well the 384-well RNA editing assay, and implement it in an HTS campaign. Aim 2 will confirm RNA editing inhibitors and determine their potency (IC50), and will assess growth inhibition of the bloodstream forms of T. brucei. Aim 3 will elucidate the nascent structure-activity relationships (SAR) of chemical scaffolds and optimize the selected hits to improve target specificity and potency of inhibitors. It will then investigate the MOA and cross-species effect of the best inhibitory compound(s), followed by scale-up to benchmark their ADME/T & PK properties. These inhibitory compound(s) will provide well-characterized chemical tool(s) or probe(s) to dissect the dynamic assembly of the editosome proteins and preferential targets that may serve as new therapeutic scaffolds against trypanosomatid pathogens.